PROJECT SUMMARY Cutaneous leishmaniasis, brought about by infection with the intracellular parasite leishmania, exhibits a spectrum of clinical manifestations, ranging from single healing lesions to severe chronic lesions, and including both disseminated and mucosal lesions, all of which can be disfiguring and/or resistant to drug treatment. Lesion resolution requires parasite control, modulation of pathologic inflammatory responses, and initiation of wound healing responses. We recently discovered that leishmania infection, in murine models and in humans, results in specific alterations to the skin microbiome, or dysbiosis, and that these changes contribute to the immunopathologic response associated with infection. In mice, the alterations occurred in a severity-dependent manner, with non-healing cutaneous leishmaniasis lesions characterized by Streptococcus spp. and resolving lesions characterized by Staphylococcus spp. Leishmania-induced dysbiosis was transmissible to non-infected cage-mates, allowing us to show that pre-existing dysbiosis results in more severe disease following an insult. In humans, preliminary gene transcriptional studies show that cutaneous leishmaniasis lesions with measurable Streptococcus reads exhibited increased IL-1? expression and were enriched for differentially expressed genes associated with cytotoxicity. This is in line with previous work where we identified a pathway leading to severe inflammation that is initiated by exaggerated T cell cytotoxicity, leading to IL-1? production. These preliminary findings provide a platform for us to delineate the sufficiency and necessity of the skin microbiome in the development of cutaneous lesions and in the wound healing response that is essential for lesion resolution (Aim 1). Our studies indicate that dysbiotic mice exhibit changes in the immune response, and we propose to define the innate and adaptive responses associated with dysbiosis that contribute to lesion development and impair wound healing (Aim 2). Importantly, our approach is designed to maximize clinical relevance by evaluating the effect of patient bacterial isolates on lesion development and resolution in mice, and by investigating the predictive nature of the microbiome in cutaneous leishmaniasis patients (Aim 3). With these studies we will determine how the skin microbiome integrates with the immune response and the wound healing response to influence disease outcomes, which will allow us to identify potential therapies to block dysbiosis-associated pathology, promote increased wound healing, and lessen disease severity in cutaneous leishmaniasis patients.